WO2021258738A1 - Tissue culture and rapid propagation method for high-quality hippeastrum vittatum seedlings - Google Patents

Tissue culture and rapid propagation method for high-quality hippeastrum vittatum seedlings Download PDF

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WO2021258738A1
WO2021258738A1 PCT/CN2021/074959 CN2021074959W WO2021258738A1 WO 2021258738 A1 WO2021258738 A1 WO 2021258738A1 CN 2021074959 W CN2021074959 W CN 2021074959W WO 2021258738 A1 WO2021258738 A1 WO 2021258738A1
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hippeastrum
culture
medium
light
seedlings
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PCT/CN2021/074959
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French (fr)
Chinese (zh)
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曾宋君
吴坤林
房林
邓莎
李琳
周世明
张文心
曹雪莹
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中国科学院华南植物园
广东圣茵花卉园艺有限公司
东莞市圣茵城市景观农业工程研究中心
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Publication of WO2021258738A1 publication Critical patent/WO2021258738A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion

Definitions

  • the invention belongs to the technical field of plant tissue culture, and specifically relates to a method for rapid propagation of Hippeastrum high-quality seedling tissue culture.
  • Hippeastrum is a perennial herb of the Amaryllidaceae Hippeastrum (Hippeastrum). It is native to Central and South America. There are about 100 native species. However, most of the popular commercial species on the market are hybrids, and the commercial seedlings are mostly cut. It is reproduced by the ball method, but it usually takes about 10 years from the breeding of a new variety to the industrialization promotion. The breeding of Hippeastrum in my country started late, but some new varieties have been bred through hybridization and other methods. If the cutting method is adopted, it is difficult to promote the large-scale production of seedlings in the short term. The use of tissue culture technology can effectively solve the problem. With regard to this problem, large-scale production of its seedlings can be carried out in 1-2 years.
  • the purpose of the present invention is to overcome the shortcomings in the prior art and provide a method for rapid propagation of high-quality seedling tissue culture of Hippeastrum hirsutum, so as to carry out the large-scale production of high-quality seedlings of the new variety of Hippeastrum hirsutum.
  • a method for rapid propagation of Hippeastrum high-quality seedling tissue culture including the following steps:
  • a. Disinfection of explants use Hippeastrum bulbs as explants to remove the leaves and roots, cut and cut the bulb discs.
  • the bulb discs are 2-3 cm long.
  • Inoculate the adventitious bud induction medium for primary culture the culture temperature is 24-30°C
  • the light is 3000-3500lx
  • the light is 12-16 hours/day
  • the adventitious bud induction medium contains: 6-benzylpurine 5.0 per liter -10.0 mg, thidiazuron 0.5-1.0 mg, sucrose 30 g, agar 6.0-6.5 g, the balance is MS medium, pH 5.8-6.0;
  • the adventitious bud primary proliferation medium per liter contains: 6-benzylpurine 5.0-10.0 mg, thidiazuron 0.5-1.0 mg, silver nitrate 1.0-5.0 mg, sucrose 30 g, agar 6.0-6.5 g, and the rest The amount is MS medium, pH 5.8-6.0;
  • the strong seedling medium contains: 6-benzylpurine 3.0-5.0 mg, sucrose 40-60 g, agar 6.0-6.5 g, and the balance is modified MS medium ( NH4NO3 and KNO3 are changed to 1.5 times of the original, and the other components remain unchanged), pH 5.8-6.0;
  • the bulbs obtained in step d After cutting the bulbs obtained in step d, inoculate them on a proliferation medium and cultivate them at a culture temperature of 24-30°C, light 3000-3500lx, light 12-16 hours/day, and the bulbs will form clusters of buds;
  • the clumping buds can be used for rooting culture or repeat steps d and e for sub-proliferation culture;
  • the proliferation medium contains: 6-benzylpurine 5.0-10.0 mg, thidiazuron 0.5-1.0 mg, and 30 sucrose per liter.
  • G 6.0-6.5 g of agar, the balance is MS medium, pH 5.8-6.0;
  • Rooting culture cut the obtained cluster buds into single buds and inoculate them on a rooting medium for rooting.
  • the culture temperature is 24-30°C
  • the light is 3000-3500lx
  • the light is 12-16 hours/day to obtain test tube plantlets;
  • Each liter of rooting medium contains: indolebutyric acid 0.5-1.0 mg, naphthalene acetic acid 0.5-1.0 mg, sucrose 40-60 g, banana homogenate 50-100 g, agar 6.0-6.5 g, the balance is modified MS culture Base (NH4NO3 and KNO3 are changed to 1.5 times of the original, and the other components remain unchanged), pH 5.8-6.0;
  • Transplanting test-tube seedlings Transfer the test-tube seedlings to smelt the seedlings under natural light, then wash the root culture medium, and transfer them to the cultivation medium for cultivation to obtain Hippeastrum seedlings.
  • the disinfection treatment of the cut bulb disc specifically includes: soaking the cut bulb disc in 75% alcohol by volume for 10-30 seconds, and soaking it with 1.0% available chlorine hypochlorous acid solution for 8-10 minutes, Rinse with sterile water for 4-5 times, then disinfect with 0.1% mercury liter solution for 8-10 minutes, rinse with sterile water for 4-5 times, and then place in 1500-2000 times dilution of medical streptomycin and shake for 24 hours .
  • the explants are obtained by the following pretreatment method: choose adult Hippeastrum bulbs as the material, and use 50% carbendazim wettable powder 500-800 times diluent 42 days before the selection of the explants for disinfection. Water once and spray the leaves. After 7 days, water and spray the leaves with 72% agricultural streptomycin soluble powder 3000-4000 times dilution, then every 7 days, use 72% agricultural streptomycin soluble powder 3000-4000 times The diluted solution was watered once and the leaves were sprayed, and 7 days after the last watering of the 72% agricultural streptomycin soluble powder 3000-4000 times dilution, the treated Hippeastrum bulbs were selected as explants.
  • the cultivation substrate includes peat, coconut bran and perlite, and the volume ratio of the peat, coconut bran and perlite is 3:3:1.
  • Said Zhudinghong is Hippeastrum'Shengyin No.1', Hippeastrum'Shengyin No.3' or Hippeastrum'Shuangyan'.
  • the cephalosporin solution is an aqueous solution of cephalosporin with a concentration of 30-50 mg/L.
  • the MS is an internationally-used medium, and its ingredients and preparation methods can be found in the literature (Tan Wencheng, Dai Cegang editors. Ornamental plant tissue culture technology. Beijing: China Forestry Publishing House, 1991.); the improved MS medium is The NH 4 NO 3 and KNO 3 in the MS medium were changed to 1.5 times the original, and the rest of the composition remained unchanged.
  • the invention uses the bulbs of the new variety of Hippeastrum with high ornamental value as explants. After material selection and treatment and explant disinfection, the disinfection success rate can reach 90-95%.
  • the unique culture medium is used to induce, proliferate and induce clumping buds. High-quality seedlings are rapidly propagated during rooting culture and other processes, and the survival rate of transplanting can reach more than 95%. Test-tube seedlings can bloom within 1.5 to 2 years after transplanting.
  • the new Hippeastrum seedlings produced by the method of the present invention are consistent Good, high genetic stability, good seedling quality, early flowering, etc., realize the large-scale production of high-quality seedlings of Hippeastrum, and can provide an effective way to meet the needs of the high-quality seedling market of new varieties of Hippeastrum.
  • the technology of the invention is practical and has high application value. Only simple plant tissue culture equipment is needed to implement the invention.
  • Example 1 Tissue culture of Hippeastrum ‘Shengyin No. 1’
  • Hippeastrum'Shengyin No.1' was selected in 2008 using H.Sandra Otway as the female parent and H.Red Peacock as the male parent. A new variety of Hippeastrum that was approved by Guangdong Province in the year. Choose adult Shengyin No. 1 Hippeastrum bulbs as the material. 42 days before the explants are selected for disinfection, first water with 50% carbendazim wettable powder 500 times dilution and spray the leaves, 7 days later, use 72% for agricultural use The 3000-fold dilution of streptomycin soluble powder was watered once and the leaves were sprayed.
  • the culture temperature is 24°C
  • the light is 3000lx
  • the light is 16 hours/day.
  • Adventitious bud induction medium contains: 6-benzylpurine (6-BA) 5.0 mg, thidiazuron (TDZ) 1.0 mg, sucrose 30 g, agar 6 g, the balance is MS medium, pH 5.8.
  • the disinfection success rate of the primary culture on the surface of the medium is 90%.
  • the adventitious buds formed by the primary culture are transferred to the adventitious bud primary proliferation medium for subculture, the culture temperature is 24°C, the light is 3000lx, and the light is 16 hours/day.
  • the adventitious bud primary proliferation medium contains: 6-benzylpurine ( 6-BA) 5.0 mg, thidiazuron (TDZ) 1.0 mg, silver nitrate 1.0 mg, sucrose 30 g, agar 6 g, the balance is MS medium, pH 5.8. No contamination by endophytes. In about 45 days, each explant can form 1 cluster bud, and the proliferation multiple is low.
  • the clump buds obtained by subculture are cut into single buds and placed on a strong seedling medium for strong seedling culture.
  • the culture temperature is 24°C
  • the light is 3000lx
  • the light is 16 hours/day.
  • the strong seedling medium contains: 6-benzyl per liter Purine (6-BA) 3.0 mg, sucrose 40 g, agar 6 g, the balance is modified MS medium, pH 5.8.
  • a small bulb with a diameter of 1.0 cm will be formed at the base of a single bud after 40 days of cultivation.
  • the culture temperature is 24°C
  • the light is 3000lx
  • the light is 16 hours/day.
  • the proliferation medium contains: 6-benzylpurine (6-BA) 5.0 mg per liter , Thidiazuron (TDZ) 1.0 mg, sucrose 30 g, agar 6 g, the balance is MS medium, pH 5.8.
  • 6-BA 6-benzylpurine
  • TDZ Thidiazuron
  • sucrose 30 g sucrose 30 g
  • agar 6 g the balance is MS medium, pH 5.8.
  • Each small bulb can form 3 clusters of buds in about 50 days.
  • the culture temperature is 24°C
  • the light is 3000lx
  • the light is 16 hours/day.
  • the rooting medium contains: indole butyric acid (IBA) 0.5 mg per liter , Naphthalene acetic acid (NAA) 1.0 mg, 40 g sucrose, 100 g banana homogenate, 6 g agar, the balance is modified MS medium, pH 5.8.
  • the rooting rate is 100%, and a complete test tube plantlet with a bulb diameter of 1.0 cm can be formed after 60 days.
  • Example 2 Tissue culture of Hippeastrum'Shengyin No.3'
  • Hippeastrum'Shengyin No.3' was born in 2005 with'Pink Girl' (H.'Pink Girl') as the female parent and'H.'Blossom Peacock' (H.'Blossom Peacock') as the parent This is a new variety of Hippeastrum that was crossed and passed the approval of Guangdong Republic in 2018.
  • the culture temperature is 27°C
  • the light is 3300lx
  • the light is 14 hours/day.
  • Adventitious bud induction medium contains 7.5 mg of 6-benzylpurine (6-BA), 0.8 mg of thidiazuron (TDZ), 30 g of sucrose, and 6.5 g of agar per liter.
  • the balance is MS medium, pH 5.9.
  • the success rate of disinfection of the primary culture on the surface of the medium was 93%.
  • the adventitious buds formed by the primary culture were transferred to the adventitious bud primary proliferation medium for subculture, the culture temperature was 27°C, the light was 3300lx, and the light was 14 hours/day.
  • Each liter of the adventitious bud primary proliferation medium contained: 6-benzylpurine ( 6-BA) 7.5 mg, thidiazuron (TDZ) 0.8 mg, silver nitrate 3.0 mg, sucrose 30 g, agar 6.5 g, the balance is MS medium, pH 5.9. No contamination by endophytes.
  • 6-BA 6-benzylpurine
  • TDZ thidiazuron
  • Each explant can form 1.5 cluster buds in about 45 days, and the proliferation multiple is low.
  • the culture temperature is 27°C
  • the light is 3300lx
  • the light is 14 hours/day.
  • the strong seedling medium contains: 6-benzyl per liter Purine (6-BA) 4.0 mg, sucrose 50 g, agar 6.5 g, the balance is modified MS medium, pH 5.9. A small bulb with a diameter of 1.2 cm will be formed at the base of a single bud after 45 days of cultivation.
  • the culture temperature is 27°C
  • the light is 3300lx
  • the light is 14 hours/day.
  • the proliferation medium contains: 6-benzylpurine (6-BA) 7.5 mg per liter , Thidiazuron (TDZ) 0.8 mg, sucrose 30 g, agar 6.5 g, the balance is MS medium, pH 5.9.
  • 6-BA 6-benzylpurine
  • TDZ Thidiazuron
  • sucrose 30 g sucrose 30 g
  • agar 6.5 g agar 6.5 g
  • the balance is MS medium, pH 5.9.
  • Each bulb can form 3.5 clusters of buds in about 50 days.
  • the culture temperature is 27°C
  • the light is 3300lx
  • the light is 14 hours/day.
  • the rooting medium contains: indole butyric acid (IBA) 0.8 mg per liter , Naphthaleneacetic acid (NAA) 0.8 mg, sucrose 50 g, banana homogenate 50 g, agar 6.5 g, the balance is modified MS medium, pH 5.9.
  • the rooting rate is 100%, and a complete test tube plantlet with a bulb diameter of 1.2 cm can be formed after 60 days.
  • Hippeastrum'Shuangyan' was selected in 2009 using the introduced Minerva (H.Minerva) as the female parent and Sandra Otway (H.Sandra Otway) as the male parent.
  • Minerva H.Minerva
  • Sandra Otway H.Sandra Otway
  • adventitious bud induction medium contains 10.0 mg 6-benzylpurine (6-BA), 0.5 mg thidiazuron (TDZ), 30 g sucrose, 6.5 g agar, and the balance is MS medium, pH 6.0.
  • the disinfection success rate of the primary culture on the surface of the medium is 95%.
  • the adventitious buds formed by the primary culture are transferred to the adventitious bud primary proliferation medium for subculture, the culture temperature is 30°C, the light is 3500lx, and the light is 12 hours/day.
  • Each liter of the adventitious bud primary proliferation medium contains: 6-benzylpurine ( 6-BA) 10.0 mg, thidiazuron (TDZ) 0.5 mg, silver nitrate 5.0 mg, sucrose 30 g, agar 6.5 g, the balance is MS medium, pH 6.0. No contamination by endophytes.
  • 6-BA 6-benzylpurine
  • TDZ thidiazuron
  • Each explant can form 2 clusters of buds in about 45 days, and the proliferation multiple is low.
  • the culture temperature is 30°C
  • the light is 3500lx
  • the light is 12 hours/day.
  • the strong seedling medium contains: 6-benzyl per liter Purine (6-BA) 5.0 mg, sucrose 60 g, agar 6.5 g, the balance is modified MS medium, pH 6.0.
  • a small bulb with a diameter of 1.5 cm will be formed at the base of a single bud after 50 days of cultivation.
  • the culture temperature is 30°C
  • the light is 3500lx
  • the light is 12 hours/day.
  • the proliferation medium contains: 6-benzylpurine (6-BA) 10.0 mg per liter , Thidiazuron (TDZ) 0.5 mg, sucrose 30 g, agar 6.5 g, the balance is MS medium, pH 6.0.
  • 6-BA 6-benzylpurine
  • TDZ Thidiazuron
  • sucrose 30 g sucrose 30 g
  • agar 6.5 g agar 6.5 g
  • the balance is MS medium, pH 6.0.
  • Each bulb can form 4 clusters of buds in about 50 days.
  • the culture temperature is 30°C
  • the light is 3500lx
  • the light is 12 hours/day.
  • the rooting medium contains: Indolebutyric acid (IBA) 1.0 mg per liter , Naphthalene acetic acid (NAA) 0.5 mg, sucrose 60 g, banana homogenate 100 g, agar 6.5 g, the balance is modified MS medium, pH 6.0.
  • the rooting rate is 100%, and a complete test tube seedling with a bulb diameter of 1.5 cm can be formed after 60 days.

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Abstract

Provided is a tissue culture and rapid propagation method for high-quality Hippeastrum vittatum seedlings. According to the method, Hippeastrum vittatum bulbs are used as explants, material selection, pretreatment and explant disinfection are performed, and the disinfection success rate can reach 90%-95%; and high-quality seedlings are rapidly propagated by means of performing steps such as induction, propagation, and rooting culture of multiple shoots, wherein the transplanting survival rate can reach 95% or above, and test-tube seedlings can flower after being transplanted for 1.5-2 years. The new Hippeastrum vittatum variety seedlings produced by means of the method have the characteristics of being good in terms of consistency, high in hereditary stability, good in seedling quality, early to flower etc., and large-scale production of high-quality Hippeastrum vittatum seedlings is achieved.

Description

一种朱顶红优质种苗组织培养快速繁殖方法A method for rapid propagation of Hippeastrum high-quality seedling tissue culture 技术领域：Technical field:
本发明属于植物组织培养技术领域，具体涉及一种朱顶红优质种苗组织培养快速繁殖方法。The invention belongs to the technical field of plant tissue culture, and specifically relates to a method for rapid propagation of Hippeastrum high-quality seedling tissue culture.
背景技术：Background technique:
朱顶红为石蒜科朱顶红属(Hippeastrum)多年生草本植物，原产于中南美洲，原生种约有100个，但目前市场上流行的商品种多为杂交种，其商品化生产的种苗多采用切球方式繁殖而来，但从一个新品种的育成到产业化推广一般需要10年左右的时间。我国朱顶红育种起步较晚，但也通过杂交等方法选育出的一些新品种，如果采用切球方式，短期内很难进行种苗的规模化生产而进行推广，利用组织培养技术能有效地解决这个问题，1－2年就能进行其种苗的规模化生产。在植物的组织培养中，通过愈伤组织或体细胞再生体系，存在较大的变异风险，利用丛生芽增殖变异较少。以朱顶红鳞茎为外植体时，存在内生菌而去污难、增殖系数较低等难题。本发明通过独特的处理方法和培养方式及独特的培养基有效地解决了这个问题。Hippeastrum is a perennial herb of the Amaryllidaceae Hippeastrum (Hippeastrum). It is native to Central and South America. There are about 100 native species. However, most of the popular commercial species on the market are hybrids, and the commercial seedlings are mostly cut. It is reproduced by the ball method, but it usually takes about 10 years from the breeding of a new variety to the industrialization promotion. The breeding of Hippeastrum in my country started late, but some new varieties have been bred through hybridization and other methods. If the cutting method is adopted, it is difficult to promote the large-scale production of seedlings in the short term. The use of tissue culture technology can effectively solve the problem. With regard to this problem, large-scale production of its seedlings can be carried out in 1-2 years. In plant tissue culture, there is a greater risk of mutation through the callus or somatic cell regeneration system, and the use of cluster buds to multiply and mutate less. When Hippeastrum bulbs are used as explants, there are problems such as difficulty in decontamination due to endophytic bacteria and low multiplication coefficient. The invention effectively solves this problem through a unique processing method, a culture method and a unique culture medium.
发明内容：Summary of the invention:
本发明的目的是克服现有技术中的不足，提供一种朱顶红优质种苗组织培养快速繁殖方法，从而进行朱顶红新品种优良株系的优质种苗的规模化生产。The purpose of the present invention is to overcome the shortcomings in the prior art and provide a method for rapid propagation of high-quality seedling tissue culture of Hippeastrum hirsutum, so as to carry out the large-scale production of high-quality seedlings of the new variety of Hippeastrum hirsutum.
本发明是通过以下技术方案实现的：一种朱顶红优质种苗组织培养快速繁殖方法，包括以下步骤：The present invention is realized through the following technical scheme: A method for rapid propagation of Hippeastrum high-quality seedling tissue culture, including the following steps:
a、外植体消毒：将朱顶红鳞茎作为外植体去除叶片和根系，将鳞茎盘切下并切割，鳞 茎盘带长度为2-3厘米的鳞茎，将切割后的鳞茎盘进行消毒处理后，接种到不定芽诱导培养基上进行初代培养，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，所述的不定芽诱导培养基每升含有：6-苄基嘌呤5.0-10.0毫克、噻苯隆0.5-1.0毫克、蔗糖30克、琼脂6.0-6.5克，余量为MS培养基，pH 5.8-6.0；a. Disinfection of explants: use Hippeastrum bulbs as explants to remove the leaves and roots, cut and cut the bulb discs. The bulb discs are 2-3 cm long. After the cut bulb discs are disinfected, Inoculate the adventitious bud induction medium for primary culture, the culture temperature is 24-30℃, the light is 3000-3500lx, the light is 12-16 hours/day, the adventitious bud induction medium contains: 6-benzylpurine 5.0 per liter -10.0 mg, thidiazuron 0.5-1.0 mg, sucrose 30 g, agar 6.0-6.5 g, the balance is MS medium, pH 5.8-6.0;
b、不定芽诱导：将初代培养的鳞茎盘转入新的所述的不定芽诱导培养基上继续初代培养，培养基表面加头孢霉素溶液，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，鳞茎盘基部形成不定芽；b. Adventitious bud induction: transfer the bulb plate of primary culture to the new adventitious bud induction medium to continue primary culture, add cephalosporin solution to the surface of the medium, culture temperature 24-30°C, light 3000-3500lx, 12-16 hours/day of light, adventitious buds are formed at the base of the bulb disc;
c、不定芽初代增殖：将初代培养形成的不定芽转入不定芽初代增殖培养基上继代培养，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，获得丛生芽；所述的不定芽初代增殖培养基每升含有：6-苄基嘌呤5.0-10.0毫克、噻苯隆0.5-1.0毫克、硝酸银1.0-5.0毫克、蔗糖30克、琼脂6.0-6.5克，余量为MS培养基，pH 5.8-6.0；c. Proliferation of adventitious buds in the first generation: transfer the adventitious buds formed in the first generation culture to the adventitious bud proliferation medium for subculture, culture temperature 24-30℃, light 3000-3500lx, light 12-16 hours/day to obtain clump buds The adventitious bud primary proliferation medium per liter contains: 6-benzylpurine 5.0-10.0 mg, thidiazuron 0.5-1.0 mg, silver nitrate 1.0-5.0 mg, sucrose 30 g, agar 6.0-6.5 g, and the rest The amount is MS medium, pH 5.8-6.0;
d、不定芽壮苗培养：将继代培养获得的丛生芽切割成单芽后放入壮苗培养基上进行壮苗培养，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，单芽基部形成鳞茎；所述的壮苗培养基每升含有：6-苄基嘌呤3.0-5.0毫克、蔗糖40-60克、琼脂6.0-6.5克，余量为改良MS培养基(NH4NO3和KNO3改变为原来的1.5倍，其余成份不变)，pH 5.8-6.0；d. Adventitious bud and strong seedling culture: cut the clump buds obtained by subculture into single buds and put them on the strong seedling medium for strong seedling culture, the culture temperature is 24-30℃, the light is 3000-3500lx, the light is 12-16 hours /Day, the base of a single bud forms a bulb; the strong seedling medium contains: 6-benzylpurine 3.0-5.0 mg, sucrose 40-60 g, agar 6.0-6.5 g, and the balance is modified MS medium ( NH4NO3 and KNO3 are changed to 1.5 times of the original, and the other components remain unchanged), pH 5.8-6.0;
e、小鳞茎分切增殖：将步骤d获得的鳞茎切割后接种到增殖培养基上培养，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，鳞茎形成丛生芽；获得的丛生芽能用于生根培养或重复步骤d和e进行继代增殖培养；所述的增殖培养基每升含有：6-苄基嘌呤5.0-10.0毫克、噻苯隆0.5-1.0毫克、蔗糖30克、琼脂6.0-6.5克，余量为MS培养基，pH 5.8-6.0；e. Cut and proliferate small bulbs: After cutting the bulbs obtained in step d, inoculate them on a proliferation medium and cultivate them at a culture temperature of 24-30°C, light 3000-3500lx, light 12-16 hours/day, and the bulbs will form clusters of buds; The clumping buds can be used for rooting culture or repeat steps d and e for sub-proliferation culture; the proliferation medium contains: 6-benzylpurine 5.0-10.0 mg, thidiazuron 0.5-1.0 mg, and 30 sucrose per liter. G, 6.0-6.5 g of agar, the balance is MS medium, pH 5.8-6.0;
f、生根培养：将获得的丛生芽切成单芽后接种到生根培养基上进行生根，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，获得试管苗；所述的生根培养基每升含有：吲哚丁酸0.5-1.0毫克、萘乙酸0.5-1.0毫克、蔗糖40-60克、香蕉匀浆50-100克、琼脂6.0-6.5克，余量为改良MS培养基(NH4NO3和KNO3改变为原来的1.5倍，其余成份不变)，pH 5.8-6.0；f. Rooting culture: cut the obtained cluster buds into single buds and inoculate them on a rooting medium for rooting. The culture temperature is 24-30°C, the light is 3000-3500lx, and the light is 12-16 hours/day to obtain test tube plantlets; Each liter of rooting medium contains: indolebutyric acid 0.5-1.0 mg, naphthalene acetic acid 0.5-1.0 mg, sucrose 40-60 g, banana homogenate 50-100 g, agar 6.0-6.5 g, the balance is modified MS culture Base (NH4NO3 and KNO3 are changed to 1.5 times of the original, and the other components remain unchanged), pH 5.8-6.0;
g、试管苗移栽：将试管苗转移到自然光下炼苗，然后洗净根部的培养基，移入栽培基质中培养，获得朱顶红种苗。g. Transplanting test-tube seedlings: Transfer the test-tube seedlings to smelt the seedlings under natural light, then wash the root culture medium, and transfer them to the cultivation medium for cultivation to obtain Hippeastrum seedlings.
所述的将切割后的鳞茎盘进行消毒处理具体为：将切割后的鳞茎盘浸泡在体积分数75％酒精中10-30秒，用1.0％有效氯的次氯酸溶液浸泡8-10分钟，无菌水冲洗4-5次，再用质量分数0.1％升汞溶液消毒8-10分钟，无菌水冲洗4-5次，然后置于医用链霉素1500-2000倍稀释液中振荡24小时。The disinfection treatment of the cut bulb disc specifically includes: soaking the cut bulb disc in 75% alcohol by volume for 10-30 seconds, and soaking it with 1.0% available chlorine hypochlorous acid solution for 8-10 minutes, Rinse with sterile water for 4-5 times, then disinfect with 0.1% mercury liter solution for 8-10 minutes, rinse with sterile water for 4-5 times, and then place in 1500-2000 times dilution of medical streptomycin and shake for 24 hours .
所述的外植体是通过以下预处理方法获得的：选择成年朱顶红鳞茎为材料，在选取外植体消毒之前的42天前，先用50％多菌灵可湿性粉剂500-800倍稀释液浇灌一次并喷施叶片，7天后用72％农用链霉素可溶性粉剂3000-4000倍稀释液浇灌一次并喷施叶片，然后每过7天，用72％农用链霉素可溶性粉剂3000-4000倍稀释液浇灌一次并喷施叶片，最后1次浇灌72％农用链霉素可溶性粉剂3000-4000倍稀释液后7天，选取处理过的朱顶红鳞茎为外植体。The explants are obtained by the following pretreatment method: choose adult Hippeastrum bulbs as the material, and use 50% carbendazim wettable powder 500-800 times diluent 42 days before the selection of the explants for disinfection. Water once and spray the leaves. After 7 days, water and spray the leaves with 72% agricultural streptomycin soluble powder 3000-4000 times dilution, then every 7 days, use 72% agricultural streptomycin soluble powder 3000-4000 times The diluted solution was watered once and the leaves were sprayed, and 7 days after the last watering of the 72% agricultural streptomycin soluble powder 3000-4000 times dilution, the treated Hippeastrum bulbs were selected as explants.
所述的栽培基质包括泥炭、椰糠和珍珠岩，所述的泥炭、椰糠和珍珠岩的体积比为3：3：1。The cultivation substrate includes peat, coconut bran and perlite, and the volume ratio of the peat, coconut bran and perlite is 3:3:1.
所述的朱顶红为圣茵1号朱顶红(Hippeastrum‘Shengyin No.1’)、圣茵3号朱顶红(Hippeastrum‘Shengyin No.3’)或双艳朱顶红(Hippeastrum‘Shuangyan’)。Said Zhudinghong is Hippeastrum'Shengyin No.1', Hippeastrum'Shengyin No.3' or Hippeastrum'Shuangyan'.
优选，所述的头孢霉素溶液为浓度为30-50毫克/升的头孢霉素水溶液。Preferably, the cephalosporin solution is an aqueous solution of cephalosporin with a concentration of 30-50 mg/L.
所述的MS为国际通用的培养基，其成分和配制方法参看文献(谭文澄、戴策刚主编.观赏植物组织培养技术.北京：中国林业出版社，1991.)；所述的改良MS培养基是将MS培养基中的NH 4NO 3和KNO 3改变为原来的1.5倍，其余成份不变。 The MS is an internationally-used medium, and its ingredients and preparation methods can be found in the literature (Tan Wencheng, Dai Cegang editors. Ornamental plant tissue culture technology. Beijing: China Forestry Publishing House, 1991.); the improved MS medium is The NH 4 NO 3 and KNO 3 in the MS medium were changed to 1.5 times the original, and the rest of the composition remained unchanged.
本发明采用观赏价值高的朱顶红新品种鳞茎为外植体，经过材料选择和处理、外植体消毒，消毒成功率可达90-95％，采用独特的培养基进行丛生芽的诱导、增殖和生根培养等过程进行优质种苗快速繁殖，移栽的成活率均可达95％以上，试管苗移栽1.5至2年就可开花，本发明的方法生产出的朱顶红新品种种苗具有一致性好，遗传稳定性高，种苗品质好、开花早等特点，实现了朱顶红优质种苗的规模化生产，能为满足朱顶红新品种优质种苗市场的需要提供一条有效的途径。The invention uses the bulbs of the new variety of Hippeastrum with high ornamental value as explants. After material selection and treatment and explant disinfection, the disinfection success rate can reach 90-95%. The unique culture medium is used to induce, proliferate and induce clumping buds. High-quality seedlings are rapidly propagated during rooting culture and other processes, and the survival rate of transplanting can reach more than 95%. Test-tube seedlings can bloom within 1.5 to 2 years after transplanting. The new Hippeastrum seedlings produced by the method of the present invention are consistent Good, high genetic stability, good seedling quality, early flowering, etc., realize the large-scale production of high-quality seedlings of Hippeastrum, and can provide an effective way to meet the needs of the high-quality seedling market of new varieties of Hippeastrum.
本发明技术切实可行，应用价值高。实施该发明只需有简单的植物组织培养设备即可进行。The technology of the invention is practical and has high application value. Only simple plant tissue culture equipment is needed to implement the invention.
具体实施方式：detailed description:
以下实施例是对本发明进一步说明，而不是对本发明的限制。The following examples are to further illustrate the present invention, but not to limit the present invention.
实施例1：圣茵1号朱顶红(Hippeastrum‘Shengyin No.1’)的组织培养Example 1: Tissue culture of Hippeastrum ‘Shengyin No. 1’
(1)材料选择和处理(1) Material selection and processing
圣茵1号朱顶红(Hippeastrum‘Shengyin No.1’)是2008年以本菲卡朱顶红(H.Sandra Otway)为母本、红孔雀朱顶红(H.Red Peacock)为父本进行杂交选育出来并于2020年通过广东省审定的朱顶红新品种。选择成年圣茵1号朱顶红鳞茎为材料，在选取外植体消毒之前的42天前，先用50％多菌灵可湿性粉剂500倍稀释液浇灌一次并喷施叶片，7天后用72％ 农用链霉素可溶性粉剂3000倍稀释液浇灌一次并喷施叶片。然后每过7天，用72％农用链霉素可溶性粉剂3000倍稀释液浇灌一次并喷施叶片，最后1次浇灌72％农用链霉素可溶性粉剂3000倍稀释液后7天，选取处理过的鳞茎为外植体。Hippeastrum'Shengyin No.1' was selected in 2008 using H.Sandra Otway as the female parent and H.Red Peacock as the male parent. A new variety of Hippeastrum that was approved by Guangdong Province in the year. Choose adult Shengyin No. 1 Hippeastrum bulbs as the material. 42 days before the explants are selected for disinfection, first water with 50% carbendazim wettable powder 500 times dilution and spray the leaves, 7 days later, use 72% for agricultural use The 3000-fold dilution of streptomycin soluble powder was watered once and the leaves were sprayed. Then every 7 days, use 72% agricultural streptomycin soluble powder 3000 times dilution to water once and spray the leaves, the last 7 days after the last watering 72% agricultural streptomycin soluble powder 3000 times dilution, select the treated The bulb is an explant.
(2)外植体消毒(2) Disinfection of explants
在超净工作台上将鳞茎外植体去除叶片和根系后用体积分数75％酒精棉擦拭干净后将鳞茎盘切下，分成8块，鳞茎盘带长度为3厘米的鳞茎。然后将切割后的鳞茎盘浸泡在体积分数75％酒精10秒，用1.0％有效氯的次氯酸溶液浸泡10分钟，无菌水冲洗4次，再用质量分数0.1％升汞溶液消毒10分钟，无菌水冲洗5次。然后置于医用链霉素1500倍稀释液中在摇床中振荡24小时后，接种到不定芽诱导培养基上进行初代培养，培养温度24℃，光照3000lx，光照16小时/天。不定芽诱导培养基每升含有：6-苄基嘌呤(6-BA)5.0毫克、噻苯隆(TDZ)1.0毫克、蔗糖30克、琼脂6克，余量为MS培养基，pH 5.8。培养基表面初代培养消毒成功率90％。Remove the leaves and roots of the bulb explants on an ultra-clean workbench, wipe clean with 75% alcohol cotton, and cut the bulb discs into 8 pieces. The bulb discs are 3 cm in length. Then soak the cut bulb in 75% alcohol with volume fraction for 10 seconds, soak with 1.0% available chlorine hypochlorous acid solution for 10 minutes, rinse with sterile water 4 times, and then sterilize with 0.1% mass fraction mercury solution for 10 minutes , Rinse 5 times with sterile water. Then place it in a 1500-fold dilution of medical streptomycin and shake it in a shaker for 24 hours, and then inoculate the adventitious bud induction medium for primary culture. The culture temperature is 24°C, the light is 3000lx, and the light is 16 hours/day. Adventitious bud induction medium contains: 6-benzylpurine (6-BA) 5.0 mg, thidiazuron (TDZ) 1.0 mg, sucrose 30 g, agar 6 g, the balance is MS medium, pH 5.8. The disinfection success rate of the primary culture on the surface of the medium is 90%.
(3)不定芽诱导(3) Adventitious bud induction
由于朱顶红鳞茎中存在较多的内生菌及易褐变，在初代培养30天时要转入新的上述的不定芽诱导培养基上继续初代培养，培养基表面加一层0.5mm高的30毫克/升头孢霉素(Cefotaxime Sodium)水溶液，培养温度24℃，光照3000lx，光照16小时/天，再培养25-30天左右能在外植体上鳞茎盘基部形成不定芽。Since there are more endophytes and easy browning in the bulbs of Hippeastrum, it should be transferred to the new adventitious bud induction medium mentioned above when the primary culture is 30 days, and the primary culture is continued with a layer of 30 mg 0.5mm high on the surface of the medium. /L of Cefotaxime Sodium aqueous solution, culture temperature 24°C, light 3000lx, light 16 hours/day, and then cultivated for about 25-30 days, adventitious buds can be formed on the base of the bulb disc on the explant.
(4)不定芽初代增殖(4) Proliferation of adventitious buds in the first generation
将初代培养形成的不定芽转入不定芽初代增殖培养基上继代培养，培养温度24℃，光照3000lx，光照16小时/天，不定芽初代增殖培养基每升含有：6-苄基嘌呤(6-BA)5.0毫克、 噻苯隆(TDZ)1.0毫克、硝酸银1.0毫克、蔗糖30克、琼脂6克，余量为MS培养基，pH 5.8。无内生菌污染。在45天左右每个外植体能形成1个丛生芽，增殖倍数较低。The adventitious buds formed by the primary culture are transferred to the adventitious bud primary proliferation medium for subculture, the culture temperature is 24℃, the light is 3000lx, and the light is 16 hours/day. The adventitious bud primary proliferation medium contains: 6-benzylpurine ( 6-BA) 5.0 mg, thidiazuron (TDZ) 1.0 mg, silver nitrate 1.0 mg, sucrose 30 g, agar 6 g, the balance is MS medium, pH 5.8. No contamination by endophytes. In about 45 days, each explant can form 1 cluster bud, and the proliferation multiple is low.
(5)不定芽壮苗培养(5) Adventitious bud and strong seedling cultivation
将继代培养获得的丛生芽切割成单芽后放入壮苗培养基上进行壮苗培养，培养温度24℃，光照3000lx，光照16小时/天，壮苗培养基每升含有：6-苄基嘌呤(6-BA)3.0毫克、蔗糖40克、琼脂6克，余量为改良MS培养基，pH 5.8。培养40天单芽基部会形成直径1.0厘米的小鳞茎。The clump buds obtained by subculture are cut into single buds and placed on a strong seedling medium for strong seedling culture. The culture temperature is 24℃, the light is 3000lx, and the light is 16 hours/day. The strong seedling medium contains: 6-benzyl per liter Purine (6-BA) 3.0 mg, sucrose 40 g, agar 6 g, the balance is modified MS medium, pH 5.8. A small bulb with a diameter of 1.0 cm will be formed at the base of a single bud after 40 days of cultivation.
(6)小鳞茎分切增殖(6) Cut and proliferate small bulbs
将每个小鳞茎切取4等份接种到增殖培养基上培养，培养温度24℃，光照3000lx，光照16小时/天，增殖培养基每升含有：6-苄基嘌呤(6-BA)5.0毫克、噻苯隆(TDZ)1.0毫克、蔗糖30克、琼脂6克，余量为MS培养基，pH 5.8。50天左右每个小鳞茎能形成3个丛生芽。Cut 4 aliquots of each small bulb and inoculate them on the proliferation medium. The culture temperature is 24℃, the light is 3000lx, and the light is 16 hours/day. The proliferation medium contains: 6-benzylpurine (6-BA) 5.0 mg per liter , Thidiazuron (TDZ) 1.0 mg, sucrose 30 g, agar 6 g, the balance is MS medium, pH 5.8. Each small bulb can form 3 clusters of buds in about 50 days.
(7)继代增殖(7) Subsequent proliferation
将小鳞茎形成的丛生芽切成单芽后重复步骤(5)和(6)进行增殖培养，能获得大量丛生芽。After cutting the cluster buds formed by the small bulbs into single buds, repeat steps (5) and (6) for multiplication and culture, and a large number of cluster buds can be obtained.
(8)生根培养(8) Rooting culture
将增殖获得的丛生芽切成单芽后接种到生根培养基上进行生根，培养温度24℃，光照3000lx，光照16小时/天，生根培养基每升含有：吲哚丁酸(IBA)0.5毫克、萘乙酸(NAA)1.0毫克、蔗糖40克、香蕉匀浆100克、琼脂6克，余量为改良MS培养基，pH 5.8。生根率100％，60天后可以形成鳞茎直径1.0厘米的完整试管苗。Cut the cluster buds obtained by proliferation into single buds and then inoculate them on a rooting medium for rooting. The culture temperature is 24℃, the light is 3000lx, and the light is 16 hours/day. The rooting medium contains: indole butyric acid (IBA) 0.5 mg per liter , Naphthalene acetic acid (NAA) 1.0 mg, 40 g sucrose, 100 g banana homogenate, 6 g agar, the balance is modified MS medium, pH 5.8. The rooting rate is 100%, and a complete test tube plantlet with a bulb diameter of 1.0 cm can be formed after 60 days.
(9)试管苗移栽(9) Transplanting test tube seedlings
将具5-6厘米高完整植株的培养瓶转移到具自然光的温室中炼苗5天，然后将其从玻璃瓶中取出，洗净根部的培养基，移入泥炭：椰糠：珍珠岩体积比为3：3：1的混合基质中，保持适当通风和足够的湿度，移栽的成活率均可达95％，试管苗移栽2年就可开花。Transfer the culture bottle with intact plants with a height of 5-6 cm to a greenhouse with natural light for 5 days to refine the seedlings, then remove them from the glass bottle, wash the root culture medium, and transfer it to the volume ratio of peat: coconut bran: perlite In a 3:3:1 mixed substrate, with proper ventilation and sufficient humidity, the survival rate of transplanting can reach 95%, and the test tube seedlings can bloom within 2 years of transplanting.
实施例2：圣茵3号朱顶红(Hippeastrum‘Shengyin No.3’)的组织培养Example 2: Tissue culture of Hippeastrum'Shengyin No.3'
(1)材料选择和处理(1) Material selection and processing
圣茵3号朱顶红(Hippeastrum‘Shengyin No.3’)是2005年以‘粉红少女朱顶红’(H.‘Pink Girl’)为母本、‘花孔雀朱顶红’(H.‘Blossom Peacock’)为父本进行杂交并于2018年通过广东省审定的朱顶红新品种。Hippeastrum'Shengyin No.3' was born in 2005 with'Pink Girl' (H.'Pink Girl') as the female parent and'H.'Blossom Peacock' (H.'Blossom Peacock') as the parent This is a new variety of Hippeastrum that was crossed and passed the approval of Guangdong Province in 2018.
选择成年圣茵3号朱顶红鳞茎为材料，在选取外植体消毒之前的42天前，先用50％多菌灵可湿性粉剂700倍稀释液浇灌一次并喷施叶片，7天后用72％农用链霉素可溶性粉剂3500倍稀释液浇灌一次并喷施叶片。然后每过7天，用72％农用链霉素可溶性粉剂3500倍稀释液浇灌一次并喷施叶片，最后1次浇灌72％农用链霉素可溶性粉剂3500倍稀释液后7天，选取处理过的鳞茎为外植体。Choose adult Shengyin No. 3 Hippeastrum bulbs as the material. 42 days before the explants are selected for disinfection, first water with a 700-fold dilution of 50% carbendazim wettable powder and spray the leaves, 7 days later, 72% agricultural use The 3500 times dilution of streptomycin soluble powder was watered once and the leaves were sprayed. Then every 7 days, water with 3500 times dilution of 72% agricultural streptomycin soluble powder and spray the leaves, and 7 days after the last watering of 3500 times dilution of 72% agricultural streptomycin soluble powder, select the treated The bulb is an explant.
(2)外植体消毒(2) Disinfection of explants
在超净工作台上将鳞茎外植体去除叶片和根系后用体积分数75％酒精棉擦拭干净后将鳞茎盘切下，分成8块，鳞茎盘带长度为2.5厘米的鳞茎。然后将切割后的鳞茎盘浸泡在体积分数75％酒精20秒，用1.0％有效氯的次氯酸溶液浸泡8分钟，无菌水冲洗5次，再用质量分数0.1％升汞溶液消毒9分钟，无菌水冲洗5次。然后置于医用链霉素1800倍稀释液中在摇床中振荡24小时后，接种到不定芽诱导培养基上进行初代培养，培养温度27℃，光照 3300lx，光照14小时/天。不定芽诱导培养基每升含有：6-苄基嘌呤(6-BA)7.5毫克、噻苯隆(TDZ)0.8毫克、蔗糖30克、琼脂6.5克，余量为MS培养基，pH 5.9。培养基表面初代培养消毒成功率93％。Remove the leaves and roots of the bulb explants on an ultra-clean workbench, wipe clean with 75% alcohol cotton, and cut the bulb discs into 8 pieces. The bulb discs are 2.5 cm long. Then immerse the cut bulb in 75% alcohol with volume fraction for 20 seconds, soak with 1.0% available chlorine hypochlorous acid solution for 8 minutes, rinse with sterile water for 5 times, and disinfect with 0.1% mercury liter solution by mass fraction for 9 minutes , Rinse 5 times with sterile water. Then place it in a 1800-fold dilution of medical streptomycin and shake it in a shaker for 24 hours, and then inoculate it on adventitious bud induction medium for primary culture. The culture temperature is 27°C, the light is 3300lx, and the light is 14 hours/day. Adventitious bud induction medium contains 7.5 mg of 6-benzylpurine (6-BA), 0.8 mg of thidiazuron (TDZ), 30 g of sucrose, and 6.5 g of agar per liter. The balance is MS medium, pH 5.9. The success rate of disinfection of the primary culture on the surface of the medium was 93%.
(3)不定芽诱导(3) Adventitious bud induction
由于朱顶红鳞茎中存在较多的内生菌及易褐变，在初代培养30天时要转入新的上述的不定芽诱导培养基上继续初代培养，培养基表面加一层1.0mm高的40毫克/升头孢霉素(Cefotaxime Sodium)水溶液，培养温度27℃，光照3300lx，光照14小时/天，再培养58天左右能在外植体上鳞茎盘基部形成不定芽。Since there are more endophytes and easy browning in the bulbs of Hippeastrum, it should be transferred to the new adventitious bud induction medium mentioned above after 30 days of primary culture to continue primary culture, with a layer of 40 mg 1.0 mm high on the surface of the medium /L Cefotaxime Sodium aqueous solution, culture temperature 27℃, light 3300lx, light 14 hours/day, and then cultured for about 58 days, adventitious buds can be formed on the base of the bulb disc on the explants.
(4)不定芽初代增殖(4) Proliferation of adventitious buds in the first generation
将初代培养形成的不定芽转入不定芽初代增殖培养基上继代培养，培养温度27℃，光照3300lx，光照14小时/天，不定芽初代增殖培养基每升含有：6-苄基嘌呤(6-BA)7.5毫克、噻苯隆(TDZ)0.8毫克、硝酸银3.0毫克、蔗糖30克、琼脂6.5克，余量为MS培养基，pH5.9。无内生菌污染。在45天左右每个外植体能形成1.5个丛生芽，增殖倍数较低。The adventitious buds formed by the primary culture were transferred to the adventitious bud primary proliferation medium for subculture, the culture temperature was 27℃, the light was 3300lx, and the light was 14 hours/day. Each liter of the adventitious bud primary proliferation medium contained: 6-benzylpurine ( 6-BA) 7.5 mg, thidiazuron (TDZ) 0.8 mg, silver nitrate 3.0 mg, sucrose 30 g, agar 6.5 g, the balance is MS medium, pH 5.9. No contamination by endophytes. Each explant can form 1.5 cluster buds in about 45 days, and the proliferation multiple is low.
(5)不定芽壮苗培养(5) Adventitious bud and strong seedling cultivation
将继代培养获得的丛生芽切割成单芽后放入壮苗培养基上进行壮苗培养，培养温度27℃，光照3300lx，光照14小时/天，壮苗培养基每升含有：6-苄基嘌呤(6-BA)4.0毫克、蔗糖50克、琼脂6.5克，余量为改良MS培养基，pH 5.9。培养45天单芽基部会形成直径1.2厘米的小鳞茎。Cut the clump buds obtained by subculture into single buds and put them on the strong seedling medium for strong seedling culture. The culture temperature is 27℃, the light is 3300lx, and the light is 14 hours/day. The strong seedling medium contains: 6-benzyl per liter Purine (6-BA) 4.0 mg, sucrose 50 g, agar 6.5 g, the balance is modified MS medium, pH 5.9. A small bulb with a diameter of 1.2 cm will be formed at the base of a single bud after 45 days of cultivation.
(6)小鳞茎分切增殖(6) Cut and proliferate small bulbs
将每个小鳞茎切取4等份接种到增殖培养基上培养，培养温度27℃，光照3300lx，光照 14小时/天，增殖培养基每升含有：6-苄基嘌呤(6-BA)7.5毫克、噻苯隆(TDZ)0.8毫克、蔗糖30克、琼脂6.5克，余量为MS培养基，pH 5.9。50天左右每个小鳞茎能形成3.5个丛生芽。Cut 4 aliquots of each small bulb and inoculate them on the proliferation medium. The culture temperature is 27℃, the light is 3300lx, and the light is 14 hours/day. The proliferation medium contains: 6-benzylpurine (6-BA) 7.5 mg per liter , Thidiazuron (TDZ) 0.8 mg, sucrose 30 g, agar 6.5 g, the balance is MS medium, pH 5.9. Each bulb can form 3.5 clusters of buds in about 50 days.
(7)继代增殖(7) Subsequent proliferation
将小鳞茎能形成的丛生芽切成单芽后重复步骤(5)和(6)进行增殖培养，能获得大量丛生芽。After cutting the cluster buds that can be formed by small bulbs into single buds, repeat steps (5) and (6) for multiplication and culture, and a large number of cluster buds can be obtained.
(8)生根培养(8) Rooting culture
将增殖获得的丛生芽切成单芽后接种到生根培养基上进行生根，培养温度27℃，光照3300lx，光照14小时/天，生根培养基每升含有：吲哚丁酸(IBA)0.8毫克、萘乙酸(NAA)0.8毫克、蔗糖50克、香蕉匀浆50克、琼脂6.5克，余量为改良MS培养基，pH 5.9。生根率100％，60天后可以形成鳞茎直径1.2厘米的完整试管苗。Cut the cluster buds obtained by proliferation into single buds and then inoculate them on a rooting medium for rooting. The culture temperature is 27°C, the light is 3300lx, and the light is 14 hours/day. The rooting medium contains: indole butyric acid (IBA) 0.8 mg per liter , Naphthaleneacetic acid (NAA) 0.8 mg, sucrose 50 g, banana homogenate 50 g, agar 6.5 g, the balance is modified MS medium, pH 5.9. The rooting rate is 100%, and a complete test tube plantlet with a bulb diameter of 1.2 cm can be formed after 60 days.
(9)试管苗移栽(9) Transplanting test tube seedlings
将具5-6厘米高完整植株的培养瓶转移到具自然光的温室中炼苗8天，然后将其从玻璃瓶中取出，洗净根部的培养基，移入泥炭：椰糠：珍珠岩体积比为3：3：1的混合基质中，保持适当通风和足够的湿度，移栽的成活率均可达97％以上，试管苗移栽20个月就可开花。Transfer the culture bottle with intact plants with a height of 5-6 cm to a greenhouse with natural light for 8 days to refine the seedlings, then remove them from the glass bottle, wash the root culture medium, and transfer it to the volume ratio of peat: coconut bran: perlite In a 3:3:1 mixed substrate, with proper ventilation and sufficient humidity, the survival rate of transplanting can reach more than 97%, and the test-tube seedlings can bloom within 20 months of transplanting.
实施例3：双艳朱顶红(Hippeastrum‘Shuangyan’)的组织培养Example 3: Tissue culture of Hippeastrum ‘Shuangyan’
(1)材料选择和处理(1) Material selection and processing
双艳朱顶红(Hippeastrum‘Shuangyan’)是2009年以引进的米娜瓦朱顶红(H.Minerva)为母本、桑德拉奥薇朱顶红(H.Sandra Otway)为父本进行杂交选育出来并于2020年通过广 东省审定的朱顶红新品种。Hippeastrum'Shuangyan' was selected in 2009 using the introduced Minerva (H.Minerva) as the female parent and Sandra Otway (H.Sandra Otway) as the male parent. A new variety of Hippeastrum that was approved by Guangdong Province in 2020.
选择成年双艳朱顶红鳞茎为材料，在选取外植体消毒之前的42天前，先用50％多菌灵可湿性粉剂800倍稀释液浇灌一次并喷施叶片，7天后用72％农用链霉素可溶性粉剂4000倍稀释液浇灌一次并喷施叶片。然后每过7天，用72％农用链霉素可溶性粉剂4000倍稀释液浇灌一次并喷施叶片，最后1次浇灌72％农用链霉素可溶性粉剂4000倍稀释液后7天，选取处理过的鳞茎为外植体。Choose adult Shuangyan Hippeastrum bulbs as the material. 42 days before the explants are selected for disinfection, first water with 50% carbendazim WP 800 times dilution and spray the leaves, 7 days later, 72% agricultural streptomyces The soluble powder 4000 times diluted solution was watered once and the leaves were sprayed. Then every 7 days, use 72% agricultural streptomycin soluble powder 4000 times dilution to water once and spray the leaves, the last 7 days after the last watering 72% agricultural streptomycin soluble powder 4000 times dilution, select the treated The bulb is an explant.
(2)外植体消毒(2) Disinfection of explants
在超净工作台上将鳞茎外植体去除叶片和根系后用体积分数75％酒精棉擦拭干净后将鳞茎盘切下，分成16块，鳞茎盘带长度为2厘米的鳞茎。然后将切割后的鳞茎盘浸泡在体积分数75％酒精30秒，用1.0％有效氯的次氯酸溶液浸泡10分钟，无菌水冲洗5次，再用质量分数0.1％升汞溶液消毒8分钟，无菌水冲洗4次。然后置于医用链霉素2000倍稀释液中在摇床中振荡24小时后，接种到不定芽诱导培养基上进行初代培养，培养温度30℃，光照3500lx，光照12小时/天。不定芽诱导培养基每升含有：6-苄基嘌呤(6-BA)10.0毫克、噻苯隆(TDZ)0.5毫克、蔗糖30克、琼脂6.5克，余量为MS培养基，pH 6.0。培养基表面初代培养消毒成功率95％。Remove the leaves and roots of the bulb explants on an ultra-clean workbench, wipe clean with 75% alcohol cotton, and cut the bulb discs into 16 pieces. The bulb discs are 2 cm in length. Then immerse the cut bulb in 75% alcohol with volume fraction for 30 seconds, soak with 1.0% available chlorine hypochlorous acid solution for 10 minutes, rinse with sterile water 5 times, and disinfect with 0.1% mercury liter solution for 8 minutes , Rinse 4 times with sterile water. Then placed in a 2000-fold dilution of medical streptomycin and shaken in a shaker for 24 hours, and then inoculated on adventitious bud induction medium for primary culture. The culture temperature was 30°C, the light was 3500lx, and the light was 12 hours/day. Adventitious bud induction medium contains 10.0 mg 6-benzylpurine (6-BA), 0.5 mg thidiazuron (TDZ), 30 g sucrose, 6.5 g agar, and the balance is MS medium, pH 6.0. The disinfection success rate of the primary culture on the surface of the medium is 95%.
(3)不定芽诱导(3) Adventitious bud induction
由于朱顶红鳞茎中存在较多的内生菌及易褐变，在初代培养30天时要转入新的上述的不定芽诱导培养基上继续初代培养，培养基表面加一层1.5mm高的50毫克/升头孢霉素(Cefotaxime Sodium)水溶液，培养温度30℃，光照3500lx，光照12小时/天，再培养30天左右能在外植体上鳞茎盘基部形成不定芽。Since there are more endophytes and easy browning in the bulbs of Hippeastrum hirsutum, it should be transferred to the new adventitious bud induction medium mentioned above when the primary culture is 30 days, and the primary culture should be continued with a layer of 50 mg 1.5mm high on the surface of the medium. /L Cefotaxime Sodium aqueous solution, culture temperature 30℃, light 3500lx, light 12 hours/day, and then cultivated for about 30 days, adventitious buds can be formed on the base of the bulb disc on the explants.
(4)不定芽初代增殖(4) Proliferation of adventitious buds in the first generation
将初代培养形成的不定芽转入不定芽初代增殖培养基上继代培养，培养温度30℃，光照3500lx，光照12小时/天，不定芽初代增殖培养基每升含有：6-苄基嘌呤(6-BA)10.0毫克、噻苯隆(TDZ)0.5毫克、硝酸银5.0毫克、蔗糖30克、琼脂6.5克，余量为MS培养基，pH 6.0。无内生菌污染。在45天左右每个外植体能形成2个丛生芽，增殖倍数较低。The adventitious buds formed by the primary culture are transferred to the adventitious bud primary proliferation medium for subculture, the culture temperature is 30℃, the light is 3500lx, and the light is 12 hours/day. Each liter of the adventitious bud primary proliferation medium contains: 6-benzylpurine ( 6-BA) 10.0 mg, thidiazuron (TDZ) 0.5 mg, silver nitrate 5.0 mg, sucrose 30 g, agar 6.5 g, the balance is MS medium, pH 6.0. No contamination by endophytes. Each explant can form 2 clusters of buds in about 45 days, and the proliferation multiple is low.
(5)不定芽壮苗培养(5) Adventitious bud and strong seedling cultivation
将继代培养获得的丛生芽切割成单芽后放入壮苗培养基上进行壮苗培养，培养温度30℃，光照3500lx，光照12小时/天，壮苗培养基每升含有：6-苄基嘌呤(6-BA)5.0毫克、蔗糖60克、琼脂6.5克，余量为改良MS培养基，pH 6.0。培养50天单芽基部会形成直径1.5厘米的小鳞茎。Cut the clump buds obtained by subculture into single buds and put them on the strong seedling medium for strong seedling culture. The culture temperature is 30℃, the light is 3500lx, and the light is 12 hours/day. The strong seedling medium contains: 6-benzyl per liter Purine (6-BA) 5.0 mg, sucrose 60 g, agar 6.5 g, the balance is modified MS medium, pH 6.0. A small bulb with a diameter of 1.5 cm will be formed at the base of a single bud after 50 days of cultivation.
(6)小鳞茎分切增殖(6) Cut and proliferate small bulbs
将每个小鳞茎切取4等份接种到增殖培养基上培养，培养温度30℃，光照3500lx，光照12小时/天，增殖培养基每升含有：6-苄基嘌呤(6-BA)10.0毫克、噻苯隆(TDZ)0.5毫克、蔗糖30克、琼脂6.5克，余量为MS培养基，pH 6.0。50天左右每个小鳞茎能形成4个丛生芽。Cut 4 aliquots of each small bulb and inoculate it on the proliferation medium. The culture temperature is 30℃, the light is 3500lx, and the light is 12 hours/day. The proliferation medium contains: 6-benzylpurine (6-BA) 10.0 mg per liter , Thidiazuron (TDZ) 0.5 mg, sucrose 30 g, agar 6.5 g, the balance is MS medium, pH 6.0. Each bulb can form 4 clusters of buds in about 50 days.
(7)继代增殖(7) Subsequent proliferation
将小鳞茎形成的丛生芽切成单芽后重复步骤(5)和(6)进行增殖培养，能获得大量丛生芽。After cutting the cluster buds formed by the small bulbs into single buds, repeat steps (5) and (6) for multiplication and culture, and a large number of cluster buds can be obtained.
(8)生根培养(8) Rooting culture
将增殖获得的丛生芽切成单芽后接种到生根培养基上进行生根，培养温度30℃，光照 3500lx，光照12小时/天，生根培养基每升含有：吲哚丁酸(IBA)1.0毫克、萘乙酸(NAA)0.5毫克、蔗糖60克、香蕉匀浆100克、琼脂6.5克，余量为改良MS培养基，pH 6.0。生根率100％，60天后可以形成鳞茎直径1.5厘米的完整试管苗。Cut the cluster buds obtained by proliferation into single buds and inoculate them on a rooting medium for rooting. The culture temperature is 30℃, the light is 3500lx, and the light is 12 hours/day. The rooting medium contains: Indolebutyric acid (IBA) 1.0 mg per liter , Naphthalene acetic acid (NAA) 0.5 mg, sucrose 60 g, banana homogenate 100 g, agar 6.5 g, the balance is modified MS medium, pH 6.0. The rooting rate is 100%, and a complete test tube seedling with a bulb diameter of 1.5 cm can be formed after 60 days.
(9)试管苗移栽(9) Transplanting test tube seedlings
将具5-6厘米高完整植株的培养瓶转移到具自然光的温室中炼苗10天，然后将其从玻璃瓶中取出，洗净根部的培养基，移入泥炭：椰糠：珍珠岩体积比为3：3：1的混合基质中，保持适当通风和足够的湿度，移栽的成活率均可达95％以上，试管苗移栽1.5年就可开花。Transfer the culture bottle with intact plants with a height of 5-6 cm to the greenhouse with natural light to refine the seedlings for 10 days, then take it out of the glass bottle, wash the root medium, and transfer it to the volume ratio of peat: coconut bran: perlite In a 3:3:1 mixed substrate, with proper ventilation and sufficient humidity, the survival rate of transplanting can reach more than 95%, and the test-tube seedlings can bloom within 1.5 years of transplanting.

Claims (6)

  1. 一种朱顶红优质种苗组织培养快速繁殖方法，其特征在于，包括以下步骤：A method for rapid propagation of Hippeastrum high-quality seedling tissue culture, which is characterized in that it comprises the following steps:
    a、外植体消毒：将朱顶红鳞茎作为外植体去除叶片和根系，将鳞茎盘切下并切割，鳞茎盘带长度为2-3厘米的鳞茎，将切割后的鳞茎盘进行消毒处理后，接种到不定芽诱导培养基上进行初代培养，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，所述的不定芽诱导培养基每升含有：6-苄基嘌呤5.0-10.0毫克、噻苯隆0.5-1.0毫克、蔗糖30克、琼脂6.0-6.5克，余量为MS培养基，pH 5.8-6.0；a. Disinfection of explants: use Hippeastrum bulbs as explants to remove the leaves and roots, cut and cut the bulb discs. The bulb discs are 2-3 cm long. After the cut bulb discs are disinfected, Inoculate the adventitious bud induction medium for primary culture, the culture temperature is 24-30℃, the light is 3000-3500lx, the light is 12-16 hours/day, the adventitious bud induction medium contains: 6-benzylpurine 5.0 per liter -10.0 mg, thidiazuron 0.5-1.0 mg, sucrose 30 g, agar 6.0-6.5 g, the balance is MS medium, pH 5.8-6.0;
    b、不定芽诱导：将初代培养的鳞茎盘转入新的所述的不定芽诱导培养基上继续初代培养，培养基表面加头孢霉素溶液，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，鳞茎盘基部形成不定芽；b. Adventitious bud induction: transfer the bulb plate of primary culture to the new adventitious bud induction medium to continue primary culture, add cephalosporin solution to the surface of the medium, culture temperature 24-30°C, light 3000-3500lx, 12-16 hours/day of light, adventitious buds are formed at the base of the bulb disc;
    c、不定芽初代增殖：将初代培养形成的不定芽转入不定芽初代增殖培养基上继代培养，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，获得丛生芽；所述的不定芽初代增殖培养基每升含有：6-苄基嘌呤5.0-10.0毫克、噻苯隆0.5-1.0毫克、硝酸银1.0-5.0毫克、蔗糖30克、琼脂6.0-6.5克，余量为MS培养基，pH 5.8-6.0；c. Proliferation of adventitious buds in the first generation: transfer the adventitious buds formed in the first generation culture to the adventitious bud proliferation medium for subculture, culture temperature 24-30℃, light 3000-3500lx, light 12-16 hours/day to obtain clump buds The adventitious bud primary proliferation medium per liter contains: 6-benzylpurine 5.0-10.0 mg, thidiazuron 0.5-1.0 mg, silver nitrate 1.0-5.0 mg, sucrose 30 g, agar 6.0-6.5 g, and the rest The amount is MS medium, pH 5.8-6.0;
    d、不定芽壮苗培养：将继代培养获得的丛生芽切割成单芽后放入壮苗培养基上进行壮苗培养，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，单芽基部形成鳞茎；所述的壮苗培养基每升含有：6-苄基嘌呤3.0-5.0毫克、蔗糖40-60克、琼脂6.0-6.5克，余量为改良MS培养基，pH 5.8-6.0；d. Adventitious bud and strong seedling culture: cut the clump buds obtained by subculture into single buds and put them on the strong seedling medium for strong seedling culture, the culture temperature is 24-30℃, the light is 3000-3500lx, the light is 12-16 hours /Day, the base of a single bud forms a bulb; the strong seedling medium contains 3.0-5.0 mg 6-benzylpurine, 40-60 g sucrose, 6.0-6.5 g agar, and the balance is modified MS medium per liter. pH 5.8-6.0;
    e、小鳞茎分切增殖：将步骤d获得的鳞茎切割后接种到增殖培养基上培养，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，鳞茎形成丛生芽；获得的丛生芽能用于生 根培养或重复步骤d和e进行继代增殖培养；所述的增殖培养基每升含有：6-苄基嘌呤5.0-10.0毫克、噻苯隆0.5-1.0毫克、蔗糖30克、琼脂6.0-6.5克，余量为MS培养基，pH5.8-6.0；e. Cut and proliferate small bulbs: After cutting the bulbs obtained in step d, inoculate them on a proliferation medium and cultivate them at a culture temperature of 24-30°C, light 3000-3500lx, light 12-16 hours/day, and the bulbs will form clusters of buds; The clumping buds can be used for rooting culture or repeat steps d and e for sub-proliferation culture; the proliferation medium contains: 6-benzylpurine 5.0-10.0 mg, thidiazuron 0.5-1.0 mg, and 30 sucrose per liter. G, 6.0-6.5 g of agar, the balance is MS medium, pH 5.8-6.0;
    f、生根培养：将获得的丛生芽切成单芽后接种到生根培养基上进行生根，培养温度24-30℃，光照3000-3500lx，光照12-16小时/天，获得试管苗；所述的生根培养基每升含有：吲哚丁酸0.5-1.0毫克、萘乙酸0.5-1.0毫克、蔗糖40-60克、香蕉匀浆50-100克、琼脂6.0-6.5克，余量为改良MS培养基，pH 5.8-6.0；f. Rooting culture: cut the obtained cluster buds into single buds and inoculate them on a rooting medium for rooting. The culture temperature is 24-30°C, the light is 3000-3500lx, and the light is 12-16 hours/day to obtain test tube plantlets; Each liter of rooting medium contains: indolebutyric acid 0.5-1.0 mg, naphthalene acetic acid 0.5-1.0 mg, sucrose 40-60 g, banana homogenate 50-100 g, agar 6.0-6.5 g, the balance is modified MS culture Base, pH 5.8-6.0;
    g、试管苗移栽：将试管苗转移到自然光下炼苗，然后洗净根部的培养基，移入栽培基质中培养，获得朱顶红种苗。g. Transplanting test-tube seedlings: Transfer the test-tube seedlings to smelt the seedlings under natural light, then wash the roots of the culture medium, and transfer them to the cultivation substrate for cultivation to obtain Hippeastrum seedlings.
  2. 根据权利要求1所述的朱顶红优质种苗组织培养快速繁殖方法，其特征在于，所述的将切割后的鳞茎盘进行消毒处理具体为：将切割后的鳞茎盘浸泡在体积分数75％酒精中10-30秒，用1.0％有效氯的次氯酸溶液浸泡8-10分钟，无菌水冲洗4-5次，再用质量分数0.1％升汞溶液消毒8-10分钟，无菌水冲洗4-5次，然后置于医用链霉素1500-2000倍稀释液中振荡24小时。The method of tissue culture and rapid propagation of high-quality seedlings of Hippeastrum according to claim 1, wherein the disinfection of the cut bulb disc is specifically: immersing the cut bulb disc in 75% alcohol by volume 10-30 seconds, soak with 1.0% available chlorine hypochlorous acid solution for 8-10 minutes, rinse with sterile water for 4-5 times, then disinfect with 0.1% mercury liter solution for 8-10 minutes, rinse with sterile water 4 -5 times, then placed in 1500-2000 times dilution of medical streptomycin and shaken for 24 hours.
  3. 根据权利要求1或2所述的朱顶红优质种苗组织培养快速繁殖方法，其特征在于，所述的外植体是通过以下预处理方法获得的：选择成年朱顶红鳞茎为材料，在选取外植体消毒之前的42天前，先用50％多菌灵可湿性粉剂500-800倍稀释液浇灌一次并喷施叶片，7天后用72％农用链霉素可溶性粉剂3000-4000倍稀释液浇灌一次并喷施叶片，然后每过7天，用72％农用链霉素可溶性粉剂3000-4000倍稀释液浇灌一次并喷施叶片，最后1次浇灌72％农用链霉素可溶性粉剂3000-4000倍稀释液后7天，选取处理过的朱顶红鳞茎 为外植体。The method of tissue culture and rapid propagation of high-quality Hippeastrum seedlings according to claim 1 or 2, wherein the explants are obtained by the following pretreatment method: selecting adult Hippeastrum bulbs as materials, and selecting explants 42 days before disinfection, first water with 50% carbendazim WP 500-800 times dilution and spray the leaves, 7 days later, use 72% agricultural streptomycin soluble powder 3000-4000 times dilution to water once and Spray the leaves, and then every 7 days, water with 3000-4000 times dilution of 72% agricultural streptomycin soluble powder and spray the leaves once, and then water the last time with 3000-4000 times dilution of 72% agricultural streptomycin soluble powder After 7 days, the treated Hippeastrum bulbs were selected as explants.
  4. 根据权利要求1或2所述的朱顶红优质种苗组织培养快速繁殖方法，其特征在于，所述的栽培基质包括泥炭、椰糠和珍珠岩，所述的泥炭、椰糠和珍珠岩的体积比为3：3：1。The method of tissue culture and rapid propagation of Hippeastrum high-quality seedlings according to claim 1 or 2, wherein the cultivation substrate comprises peat, coconut bran and perlite, and the volume ratio of the peat, coconut bran and perlite It is 3:3:1.
  5. 根据权利要求1或2所述的朱顶红优质种苗组织培养快速繁殖方法，其特征在于，所述的朱顶红为圣茵1号朱顶红(Hippeastrum‘Shengyin No.1’)、圣茵3号朱顶红(Hippeastrum‘Shengyin No.3’)或双艳朱顶红(Hippeastrum‘Shuangyan’)。The method of tissue culture and rapid propagation of high-quality seedlings of Hippeastrum according to claim 1 or 2, wherein the Hippeastrum is Hippeastrum'Shengyin No. 1', Hippeastrum'Shengyin No. 1', Hippeastrum's Hippeastrum 'Shengyin No.3') or Hippeastrum'Shuangyan'.
  6. 根据权利要求1或2所述的朱顶红优质种苗组织培养快速繁殖方法，其特征在于，所述的头孢霉素溶液为浓度为30-50毫克/升的头孢霉素水溶液。The method of tissue culture and rapid propagation of Hippeastrum high-quality seedlings according to claim 1 or 2, wherein the cephalosporin solution is an aqueous solution of cephalosporin with a concentration of 30-50 mg/L.
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